Dendritic cells (DCs) are essential antigen presenting cells for the initiation and control of immune responses. DCs can capture and process antigens, migrate from the periphery to a lymphoid organ, and present the antigens to resting T cells in a major histocompatibility complex (MHC)-restricted fashion. These cells are derived from bone marrow (BM) and display dendritic morphology and high mobility. The discovery of DCs as specialized antigen-presenting cells (APCs) has fueled attempts at DC-based immunization/vaccination strategies that involve loading DCs in vitro with specific antigens (Banchereau and Palucka, A. K. 2005. Nat. Rev. Immunol. 5:296-306; Figdor, et al. 2004. Nat. Med. 10:475-480). All of these attempts however, involve the labor-intensive preparation of a patient-specific therapy that includes the loading of autologous DCs ex vivo with specific antigens, which are then administered to the patient.
An alternative strategy is to utilize recombinant virus-based vectors as a mechanism to directly deliver a gene encoding a designated antigen(s) to host cells. In this instance, through induction of a desired adaptive immune response, the expressed gene product provides therapeutic benefit. There are a number of challenges however to achieving a safe and effective system. Some of these challenges include designing a vector that targets a desired set of host cells, providing a suitable delivery system, expressing a desired antigen to elicit an effective immune response and consistently manufacturing a sufficiently high titered pharmaceutical composition of the recombinant virus vector virus so that it can be utilized broadly across a designated human subject population. The latter is a particular challenge in developing laboratory scale systems into products that can be produced by the pharmaceutical industry.
In the laboratory, many lentiviral vectors are pseudotyped with the VSV-G envelope proteins. This is widely used as a model system as the VSV envelope proteins are able to target many cell types (a “pantropic” envelope), and production systems generally provide a high titre.
The envelope glycoproteins of Sindbis virus and other alphaviruses disclosed herein incorporate into the lipid bilayer of the viral particle membrane. Typically, the viral membrane (envelope) includes multiple copies of trimers of two glycoprotein heterodimers. E1 and E2, which are produced from cleavage of a single precursor protein. The precursor protein comprises, from its N- to C-terminal, the E3, E2, 6K and E1 proteins. The small E3 glycoprotein serves as a signal sequence for translocation of the E2 protein into the membrane, and is cleaved from E2 by furin or some other Ca2+-dependent serine proteinase. The 6K protein serves as a signal sequence for translocation of the E1 protein into the membrane and is then cleaved from the precursor protein.
The E1 and E2 glycoproteins each have membrane-spanning regions; E2 has an about 33 residue cytoplasmic domain whereas the cytoplasmic tail of E1 is very short (about 2 residues). Both E1 and E2 have palmitic acids attached in or near the membrane-spanning regions.
Isolates of Sindbis virus described in the art are believed to infect cells via an interaction with heparan sulfate (HS). In WO 2008/011636 a lentiviral packaging system was described in which the E3/E2 envelope fusion protein (called SVGmu) contains a number of modifications, intended to reduce binding of the protein to HS but to retain binding to and infection of DCs, via the DC-SIGN surface molecule. Though the SVGmu pseudotyped viral particles were able to selectively transduce cells expressing the DC-SIGN antigen, several aspects of the system make it unsuitable for therapeutic use. For example, the E3/E2 fusion protein displays an antigenic epitope of influenza hemagglutinin, and significantly. Sindbis virus strains with a mutation preventing correct processing of E3 from the E2 glycoprotein (so-called “pE2 mutants”), such as SVGmu, grow poorly in permissive cell lines and are severely attenuated in mouse pathogenicity.